CN109266649B

CN109266649B - Inducible promoter CDM1 promoter responsive to salt stress

Info

Publication number: CN109266649B
Application number: CN201811183462.6A
Authority: CN
Inventors: 郑敏; 许一丰; 颜嘉雯; 孙腾飞
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2018-10-11
Filing date: 2018-10-11
Publication date: 2021-03-12
Anticipated expiration: 2038-10-11
Also published as: CN109266649A

Abstract

The invention discloses an inducible promoter CDM1 promoter responding to salt stress, wherein the nucleotide sequence of the CDM1 promoter is shown as SEQ ID No.1, and the CDM1 promoter is obtained by cloning from a CDM1 gene promoter region of arabidopsis thaliana. Through the analysis of the promoter region, the promoter region contains a plurality of cis-acting elements which can participate in multiple adversity stresses. After the transgenic plants are treated by a saline culture medium, the CDM1 promoter can enhance the expression of exogenous GUS (glucuronidase) genes, so that the transgenic plants are deeply dyed, and the CDM1 promoter is proved to be obviously induced by salt. As an inducible promoter capable of being expressed in a heterologous way, the CDM1 promoter region and an upstream regulatory sequence have very important values in the aspects of genetic engineering and practical application.

Description

Inducible promoter CDM1 promoter responsive to salt stress

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an inducible promoter CDM1 promoter responding to salt stress.

Background

A promoter is a component of a gene, which is recognized by RNA polymerase and initiates transcription. The inducible promoter is different from a constitutive promoter, and can start the transcription of an exogenous gene only under the stimulation of certain signals, so that the expression product of a target gene can be accumulated in a certain space-time, the regional expression level is increased, and the stress resistance of a plant is improved. Solves the problem of food safety in transgenic application to a certain extent, and is an ideal promoter for genetic engineering breeding by applying a plant transgenic technology.

In nature, various stresses exist, which seriously affect the growth of plants, such as drought, salt damage, high temperature and the like. High-concentration salt causes physiological drought of plants, damages plant tissues and influences the absorption and metabolism of the plants on nutrients, thereby influencing the normal nutrition of the plants and causing the reduction and even death of the yield of the plants.

The promoter is an important element of a gene engineering expression vector, and the research on the promoter has very important significance for improving the production traits of crops, such as insect resistance, disease resistance, stress resistance and the like, by a gene expression regulation mechanism. Currently known inducible promoters generally include: light-induced expression gene promoters, heat-induced expression gene promoters, wound-induced expression gene promoters and the like. The obtained new promoter from plant can provide useful core elements for researching expression and regulation of plant genes and improving crops by using genetic engineering methods.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: an inducible promoter is provided which initiates transcription of a foreign gene under salt stimulation.

The technical scheme of the invention is as follows: an inducible promoter responding to salt stress, CDM1 promoter, the nucleotide sequence of CDM1 promoter is shown as SEQ ID No. 1.

A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a CDM1 promoter.

Use of a CDM promoter in transgenic plants. The application is to improve the salt tolerance of plants.

Compared with the prior art, the invention has the following beneficial effects:

the CDM1 promoter was cloned from the promoter region of the Arabidopsis CDM1 gene. Through the analysis of the promoter region, the promoter region contains a plurality of cis-acting elements which can participate in multiple adversity stresses. After the transgenic plants are treated by a saline culture medium, the CDM1 promoter can enhance the expression of exogenous GUS (glucuronidase) genes, so that the transgenic plants are deeply dyed, and the expression level of CDM1 is obviously induced by salt. As an inducible promoter capable of being expressed in a heterologous way, the CDM1 promoter region and an upstream regulatory sequence have very important values in the aspects of genetic engineering and practical application.

Drawings

FIG. 1 is a CDM1 promoter sequence analysis;

FIG. 2 is an electrophoretogram of a CDM1 promoter fragment;

FIG. 3 is an AtCDM1-GUS transgenic Arabidopsis plant; phenotype of wild type (Col-0) and AtCDM1-GUS transgenic plants. 9d large seedlings grown on 1/2MS medium as shown in the figure;

FIG. 4 is the analysis of GUS staining after salt treatment of AtCDM1-GUS transgenic Arabidopsis thaliana.

Detailed Description

1. Cloning of promoters

The CDM1 gene DNA sequence was downloaded from TAIR (https:// www.arabidopsis.org /) database, and a length of about 1.6kb upstream of ATG was selected as the CDM1 promoter region, primers were designed, and the cleavage sites were Kpn I and SalI.

The promoter amplification system was as follows (20 ul in total)

TABLE 1 primer sequences for CDM1 promoter amplification

Underlined sequences are the introduced restriction enzyme recognition sites.

The experimental results are as follows:

the amplified product is 1639bp fragment detected by 1.0% agarose gel electrophoresis, as shown in FIG. 2, and its nucleotide sequence is shown in SEQ ID No. 1.

2. Promoter sequence analysis

Using PLACE database (http://www.dna.affrc.go.jp/PLACE/) Promoter regulatory element analysis was performed.

The experimental results are as follows:

TABLE 2 summary of promoter essential elements and elements responsive to environmental stress contained in CDM1 promoter

N＝A/G/C/T,W＝A/T,R＝A/G，V＝A/C/G

3. Transgenic arabidopsis plants

The constructed binary vector containing AtCDM1-GUS is transferred into agrobacterium and then transferred into wild Arabidopsis (Col-0) by a catkin dip-dyeing method. After the seeds are mature, screening on a hygromycin-containing flat plate, wherein the binary vector contains hygromycin resistance genes, so that the plants successfully transferred into the binary vector can normally grow, transferring the screened plants onto an 1/2MS flat plate, growing for 3-5 days, transplanting, and then further identifying by using a GUS dye solution. The identification method comprises the following steps: adding a proper amount of GUS staining solution into a plant to be stained to enable the GUS staining solution to completely immerse tissues, incubating overnight at 37 ℃, gradually appearing blue along with the prolonging of the incubation time, enabling the GUS active part or site to present blue or blue spots when the expression quantity is higher, removing chlorophyll of a sample by using 70% ethanol, immersing the general sample in ethanol for 1-3h, accelerating chlorophyll decoloration by using a water bath at 50 ℃, and observing by naked eyes or an optical microscope.

And (4) analyzing results: the obtained transgenic plant is basically consistent with the wild type in phenotype (see figure 3), and leaves are obviously blue after GUS staining is carried out, so that the CDM1 promoter sequence has the promoter function and can promote the expression of exogenous genes.

4. Salt induction test

The method comprises the steps of disinfecting the surfaces of wild seeds and transgenic T3 generation seeds, then placing the seeds at 4 ℃ for vernalization for three days, putting the seeds on a 1/2MS flat plate, growing the seeds under 10000Lux illumination and 16h illumination/8 h non-illumination alternately for 5 days, and transferring the wild plants and the transgenic plants which are simultaneously on the same flat plate to the same 1/2MS and 150mM NaCl flat plate respectively for growing the seeds for 48 hours in order to reduce variable and uncontrollable factors.

Seedlings grown for 48h on the above plates were harvested and subjected to GUS staining, and the results are shown in FIG. 4. After the transgenic plants are treated by a culture medium containing salt (150mM NaCl), the CDM1 promoter can enhance the expression of exogenous GUS gene, so that the transgenic plants are deeply dyed, and the CDM1 promoter is proved to be obviously induced by the salt.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

Sequence listing

<110> university of the applicant's name southwest

<120> inducible promoter in response to salt stress CDM1 promoter

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1639

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 1

ttatgtcaac gatgcgttcg ggactgctca cagagcccat gcttcaacag agggagtcac 60

taagttcttg aagccatcag ttgctggttt ccttttgcaa aaggtgacat tttgatacaa 120

gattctttct taaattgatt attgaggaat ttagaagcac cacaagtgcc gccactatat 180

ccttgatcat gaaataattg attgattgct tgttcatctt tgtctttcca tgtaagaggt 240

ctaaactcaa ggttcaatag gtttctcaac tgcaatttgg aggagtccat tgacttgaat 300

tgagttatga gtttgaattg aatcacattt ggttatctta tatatttcgc aggagttgga 360

ctaccttgtt ggtgcggttt caaacccaaa gagaccattt gctgccattg tgggaggttc 420

caaggtctca tctaagattg gagttatcga atcgcttctt gagaaatgtg acatccttct 480

gcttggtggt ggaatgatct ttacattcta caaggcgcaa ggtctttccg ttggctcctc 540

ccttgttgaa gaagacaagc ttgaattggc tacaacactc cttgccaagg ctaaggccag 600

aggagtctct ctgttgttac caacagatgt tgtgattgct gacaagttcg ctcctgatgc 660

caacagcaag gttcgtctcc aataactatt gctaaaagct tggcattctt ggttgacaga 720

aagattgaaa aatggttttg ttttttaaga ctggatgtgt tgagggtact gagtgtaatg 780

tgaatgttgt agattgtgcc agcatcagcc attcctgatg ggtggatggg attggacatc 840

ggtccagact cggtgaaaac attcaacgaa gctctggata ccacgcagac agtcatttgg 900

aatggaccaa tgggagtttt cgagtttgaa aagtttgcaa aaggaactga ggtgcatata 960

attctctgac tcttatatac tgttagttga ctctttgttg gaactaatat gtagcatggt 1020

tgaatggcgc aggcggtagc gaataaacta gcagagctaa gcaaaaaggg agtgacaacg 1080

ataataggag gaggagactc ggtggcagca gtggagaaag tgggagtagc aggagtcatg 1140

agtcacatct ccacaggtgg tggtgccagt ttggagctct tggaaggcaa agtgcttccc 1200

ggtgtcgtcg ctcttgatga agcaacgcca gtcactgttt aacaacatct ctttattaca 1260

cctgaaggga cccccctaaa tgagtttctt cttcttattc tgttgtaaat caaagtcttt 1320

gttgtaccat caaatccata gagaggaggg actctttttg tctcaaataa aatcaaatac 1380

agtgacattc gttattttga gcaaaacatc aatcaactgc tcgaaatcat tttaaccttg 1440

agaatagttt catcctcaaa ataaattttc aagaaaatac gaggactcga aacgaaaaag 1500

gatgtaaata ttcccacgtg ccgtttcatt attggctgca agttgccaaa agtatacata 1560

cgcgcaggtg aaatggattt atctatctct aaagttgttc cttcttctct ccttcgtctc 1620

ccgtcaagct gctttagcc 1639

<210> 2

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggtaccttat gtcaacgatg cgttcg 26

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtcgacggct aaagcagctt gacgg 25

Claims

1. An inducible promoter responding to salt stress, CDM1 promoter, the nucleotide sequence of CDM1 promoter is shown as SEQ ID No. 1.

2. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a CDM1 promoter with a nucleotide sequence shown in SEQ ID No. 1.

3. Use of the CDM1 promoter of claim 1 to improve salt tolerance in transgenic plants.